Protein binding miniature proteins and uses thereof

ABSTRACT

In certain aspects, the present invention provides miniature proteins resulted from a protein scaffold such as an avian pancreatic polypeptide that can be modified by substitution of at least one amino acid residue. In other aspects, the present invention provides diagnostic and therapeutic uses of these miniature proteins.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/009,101, filed Dec. 11, 2004, which claims the benefit of U.S.provisional application 60/529,401, filed Dec. 11, 2003, the disclosuresof each of which are hereby incorporated by reference in theirentireties.

FUNDING

Work described herein was funded, in part, by National Institutes ofHealth Grant GM 59843. The United States government has certain rightsto the invention.

BACKGROUND

Biological interactions, such as protein:protein interactions,protein:nucleic acid interactions, and protein:ligand interactions areinvolved in a wide variety of processes occurring in living cells. Forexample, agonism and antagonism of receptors by specific ligands mayeffect a variety of biological processes such as gene expression,cellular differentiation and growth, enzyme activity, metabolite flowand metabolite partitioning between cellular compartments. Undesirableor inappropriate gene expression and/or cellular differentiation,cellular growth and metabolism may be attributable, at least in manycases, to biological interactions involving the binding and/or activityof proteinaceous molecules, such as transcription factors, peptidehormones, receptor molecules, and enzymes.

Peptides present potential therapeutic and prophylactic agents for manyhuman and animal diseases, biochemical disorders and adverse drugeffects, because they can interact highly specifically with othermolecules. Thus, mimetic peptides have been designed and developed basedon three dimensional protein structures. For example, many proteinsrecognize nucleic acids, other proteins or macromolecular assembliesusing a partially exposed alpha helix. Within the context of a nativeprotein fold, such alpha helices are usually stabilized by extensivetertiary interactions with residues that may be distant in primarysequence from both the alpha helix and from each other. With notableexceptions (Armstrong et al., 1993, J. Mol. Biol., 230: 284-291),removal of these tertiary interactions destabilizes the alpha helix andresults in molecules that neither fold nor function in macromolecularrecognition (Zondlo et al., 1999, J. Am. Chem. Soc., 121: 6938-6939).The ability to recapitulate or perhaps even improve on the recognitionproperties of an alpha helix within the context of a small molecule mayfind utility in the design of synthetic mimetics or inhibitors ofprotein function (Cunningham et al., 1997, Curr. Opin. Struct. Biol., 7:457-462) or new tools for proteomics research.

Proteins generally recognize each other using large and shallowcomplementary surfaces. Therefore, small proteins (miniature proteins)with well-defined three-dimensional structures and finely tunedfunctional properties are perhaps ideally suited for protein surfacerecognition and disruption of protein:protein interaction. Clearly,there is a need for developing the miniature proteins (in particular,those with high affinity and high specificity for a target molecule) astherapeutics and prophylactics.

SUMMARY OF THE INVENTION

In certain embodiments, the invention relates to an avian pancreaticpolypeptide (aPP) modified by substitution of at least one amino acidresidue, which is located within (as a component of) a type IIpolyproline (PPII) helix of the polypeptide when the polypeptide is in atertiary form. In some embodiments, the modified polypeptide contains atleast two or three substituted residues. Optionally, the residuesubstitutions can include modification of position 2 (e.g., F in SEQ IDNO:1) with other hydrophobic residues (L, I, V, A). In otherembodiments, the modified polypeptide is further modified bysubstitution of at least one amino acid residue (e.g., two residues) ofthe linker region between the PPII helix and the alpha helix domain ofthe polypeptide. The modified polypeptide of the invention is alsoreferred to as a miniature protein.

In certain cases, the substituted residue of the PPII helix is selectedfrom a site on a first protein through which the first protein interactswith a second protein. The first protein can be a known protein such as,but are not limited to, a protein that interacts with EVH1 domains.Examples of the first proteins include zyxin, vinculin, and the ActAprotein of Listeria monocytogenes. The second protein which interactswith the first protein includes but is not limited to any protein thatcontains an EVH1. For example, the second protein can be selected fromthe group consisting of: Drosophila Enabled (Ena), mammalian Mena,vasodilator stimulated phosphoprotein (VASP), Enabled/VASP-like protein(Evl), and Wiskott-Aldrich syndrome protein (WASP). In a preferredembodiment, the site on the first protein is a protein binding site(e.g., a polyproline helix). In some embodiments, the modified avianpancreatic polypeptide is capable of inhibiting the interaction betweenthe first protein and the second protein, while in other embodiments, itis capable of enhancing this interaction.

In a specific embodiment, the miniature protein of the inventionpreferentially binds to one protein selected from the group consistingof: Drosophila Enabled (Ena), mammalian Mena, vasodilator stimulatedphosphoprotein (VASP), Enabled/VASP-like protein (Evl), andWiskott-Aldrich syndrome protein (WASP), but does not bind to the otherproteins of the group.

In certain embodiments, the invention encompasses a phage-displaylibrary comprising a plurality of recombinant phage that express any ofthe aforementioned modified avian pancreatic polypeptides. In a relatedembodiment, the invention encompasses a phage-display library comprisinga plurality of recombinant phage that express a protein scaffoldmodified by substitution of at least one amino acid residue, thisresidue being exposed on a type II polyproline helix of the polypeptidewhen the polypeptide is in a tertiary form. In some cases, the proteinscaffold of the phage-display library comprises the avian pancreaticpolypeptide. The invention also encompasses an isolated phage selectedfrom the phage library of the invention.

In a specific embodiment, a miniature protein of the invention comprisesthe amino acid sequence PFPPTPPGEEAPVEDLIRFYNDLQQYLNVV (SEQ ID NO: 1).In other embodiments, the miniature protein may comprise any of thefollowing amino acid sequence: PAPPTPPGEEAPVEDLIRFYNDLQQYLNVV (SEQ IDNO: 2); PFPPLPPGEEAPVEDLIRFYNDLQQYLNVV ((SEQ ID NO: 3);PLPPTPPGEEAPVEDLIRFYNDLQQYLNVV (SEQ ID NO: 4);PFPPTPPGEELPVEDLIRFYNDLQQYLNVV (SEQ ID NO: 5). Further, the presentinvention contemplates all the variants with A substituted at each non-Aposition of the avian pancreatic polypeptide, and all the variants withsarcosine substituted at positions 1-8 of the avian pancreaticpolypeptide.

Further, the present invention provides an isolated polypeptide selectedfrom the group consisting of: (a) an isolated polypeptide comprising anyof the amino acid sequences as set forth in SEQ ID NOs: 1-5; (b) anisolated polypeptide comprising a fragment of at least twelve contiguousamino acids of any of SEQ ID NOs: 1-5; (c) an isolated polypeptidecomprising one or more amino acid substitutions in any of the amino acidsequences as set forth in SEQ ID NOs: 1-5; and (d) an isolatedpolypeptide at least 95 percent identical to any of SEQ ID NOs: 1-5.

In a related embodiment, the invention also encompasses a nucleic acidencoding any one of the aforementioned miniature polypeptides of theinvention.

In certain embodiments, the invention encompasses a method of preparinga miniprotein that modulates the interaction between a first protein anda second protein, comprising the steps of: (a) identifying at least oneamino acid residue that contributes to the binding between a firstprotein and a second protein; and (b) modifying an avian pancreaticpolypeptide by substitution of said at least one amino acid residue,such that said at least one amino acid residue is exposed on a type IIpolyproline (PPII) helix of the polypeptide when the polypeptide is in atertiary form. As used herein, the term “modulate” refers to analteration (enhancement or inhibition) in the association between twomolecular species, for example, the effectiveness of a biological agentto interact with its target by altering the characteristics of theinteraction in a competitive or non-competitive manner.

In certain embodiments, the invention further encompasses a method ofidentifying a miniprotein that modulates the interaction between a firstprotein and a second protein, comprising a step of isolating at leastone recombinant phage clone from the phage display library of theinvention that displays a protein scaffold that modulates theassociation between a first protein and a second protein.

In certain embodiments, the invention provides a method of modulating(enhancing or inhibiting) cell migration, comprising contacting a cellwith a modified polypeptide of the invention in an effective amount formodulating cell migration, wherein the modified polypeptide regulatessignaling through a protein of the Ena/VASP family (an Ena/VASPprotein). In this method, the Ena/VASP protein is preferably selectedfrom the group consisting of: Drosophila Enabled (Ena), mammalian Mena,vasodilator stimulated phosphoprotein (VASP), Enabled/VASP-like protein(Evl), and Wiskott-Aldrich syndrome protein (WASP). Optionally, themodified polypeptide binds to an EVH1 domain of the Ena/VASP protein. Apreferred cell of this method is a mammalian cell. In certain cases, themammalian cell is a tumor cell.

In certain embodiments, the invention provides a method for inhibitingtumor cell metastasis in a subject, comprising administering to asubject having or at risk of developing a metastatic cancer a modifiedpolypeptide of the invention in an effective amount for inhibiting cellmigration such that tumor cell metastasis is inhibited, wherein themodified polypeptide inhibits signaling through an Ena/VASP protein. Inthis method, the Ena/VASP protein is preferably selected from the groupconsisting of: Drosophila Enabled (Ena), mammalian Mena, vasodilatorstimulated phosphoprotein (VASP), Enabled/VASP-like protein (Evl), andWiskott-Aldrich syndrome protein (WASP). Optionally, the modifiedpolypeptide binds to an EVH1 domain of the Ena/VASP protein. A preferredsubject of this method is a mammal, for example, a human.

In certain embodiments, the invention provides a method of modulating(enhancing or inhibiting) growth of a neuronal cell, comprisingcontacting a neuronal cell with a modified polypeptide of the inventionin an effective amount for modulating growth of the neuronal cell,wherein the modified polypeptide regulates signaling through an Ena/VASPprotein. In this method, the Ena/VASP protein is preferably selectedfrom the group consisting of: Drosophila Enabled (Ena), mammalian Mena,vasodilator stimulated phosphoprotein (VASP), Enabled/VASP-like protein(Evl), and Wiskott-Aldrich syndrome protein (WASP). Optionally, themodified polypeptide binds to an EVH1 domain of the Ena/VASP protein.

In certain embodiments, the invention provides a method of inhibitingneurodegeneration in a subject, comprising administering to a subject atrisk of a neurodegeneration disorder a modified polypeptide of theinvention in an amount effective to prevent neurodegeneration, whereinthe modified polypeptide regulates signaling through an Ena/VASPprotein. In this method, the Ena/VASP protein is preferably selectedfrom the group consisting of: Drosophila Enabled (Ena), mammalian Mena,vasodilator stimulated phosphoprotein (VASP), Enabled/VASP-like protein(Evl), and Wiskott-Aldrich syndrome protein (WASP). Optionally, themodified polypeptide binds to an EVH1 domain of the Ena/ASP protein. Avariety of neurodegenerative disorders can be treated by this method,such as Down Syndrome; Parkinson's disease; amyotrophic lateralsclerosis (ALS), stroke, direct trauma, Huntington's disease, epilepsy,ALS-Parkinsonism-dementia complex; progressive supranuclear palsy;progressive bulbar palsy, spinomuscular atrophy, cerebral amyloidosis,Pick's atrophy, Retts syndrome; Wilson's disease, Striatonigraldegeneration, corticobasal ganglionic degeneration; dentatorubralatrophy, olivo-pontocerebellar atrophy, paraneoplastic cerebellardegeneration; Tourettes syndrome, hypoglycemia; hypoxia;Creutzfeldt-Jakob disease; and Korsakoffs syndrome.

BRIEF DESCRIPTION OF THE DRAWINGS

The file of this patent contains at least one drawing executed in color.Copies of this patent with color drawing(s) will be provided by thePatent and Trademark Office upon request and payment of the necessaryfee.

FIG. 1A shows protein grafting of the FP4 epitope on a miniature proteinscaffold.

FIG. 1B shows sequences of peptides and miniature proteins described inthis work. Residues important for aPP folding are in blue or yellow,residues important for Mena₁₋₁₁₂ recognition are in red. These aminoacid residues are shown in duplicate in black font in the lower panel ofFIG. 1B.

FIG. 1C is a spectra showing the mean residue ellipticity (Θ_(MRE)) of 5μM pGolemi at 25° C.

FIG. 1D shows the temperature dependence of the Θ_(MRE) at 222 nm.

FIG. 1E shows amino acid sequences of four additional miniature proteinsof the invention.

FIGS. 2A-2D show interactions between EVH1 domains, miniature proteinsand peptides measured by fluorescence perturbation (A, C, and D) orfluorescence polarization (B). FIG. 2A shows binding of pGolemi(magenta), ActA₁₁ (red) or PPII7 (yellow). FIGS. 2B and 2C show bindingof pGolemi^(Flu) (25 nM, B) or pGolemi (C) to Mena₁₋₁₁₂ (circle),VASP₁₋₁₁₅ (triangle) or Evl₁₋₁₁₅ (square). FIG. 1D shows binding ofActA₁₁ to Mena₁₋₁₁₂ (circle), VASP₁₋₁₁₅ (triangle) or Evl₁₋₁₁₅ (square).Fraction bound refers to fraction of EVH1 domain bound (A, C, D) orpGolemi^(Flu) (B).

FIG. 3 shows median speed of L. monocytogenes observed in the absence(white) or presence of pGolemi (purple) and ActA₁₁ (red). Errors barsshow the intraquartile range.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is based at least in part on protein grafting, anapproach to protein minimization that was successfully developed byApplicants. The protein grafting approach has been used for theidentification of highly functional miniature proteins by stabilizationof α-helical binding epitopes on a protein scaffold (Zondlo et al.,1999, J. Am. Chem. Soc., 121:6938-6939; Chin et al., 2001, Bioorg. Med.Chem. Lett., 11:1501-1505; Chin et al., 2001, J. Am. Chem. Soc.,123:2929-2930; Chin et al., 2001, Angew Chem. Int. Ed. Engl., 2001,40:3806-3809; Montclare et al., 2003, J. Am. Chem. Soc., 125:3416-3417;Rutledge et al., 2003, J. Am. Chem. Soc., 125:14336-47). In thesemethods, protein grafting involves removing residues required formolecular recognition from their native alpha helical context andgrafting them on the scaffold provided by small yet stable proteins.

Numerous researchers have engineered protein scaffolds to presentbinding residues on a relatively small peptide carrier. These scaffoldsare small polypeptides onto which residues critical for binding to aselected target can be grafted. The grafted residues are arranged inparticular positions such that the spatial arrangement of these residuesmimics that which is found in the native protein. These scaffoldingsystems are commonly referred to as miniproteins (miniature proteins). Acommon feature is that the binding residues are known before theminiprotein is constructed.

Examples of these miniproteins include the thirty-seven amino acidprotein charybdotoxin (Vita et al., 1995, Proc. Natl. Acad. Sci. USA,92: 6404-6408; Vita et al., 1998, Biopolymers, 47: 93-100) and thethirty-six amino acid protein, avian pancreatic peptide (Zondlo et al.,1999, Am. Chem. Soc., 121: 6938-6939). Avian pancreatic polypeptide(aPP) is a polypeptide in which residues fourteen through thirty-twoform an alpha helix stabilized by hydrophobic contacts with anN-terminal type II polyproline (PPII) helix formed by residues onethrough eight. Because of its small size and stability, aPP is anexcellent scaffold for protein grafting of alpha helical recognitionepitopes (Zondlo et al., 1999, Am. Chem. Soc., 121: 6938-6939).

Miniature Proteins

The present invention provides engineered miniature proteins thatassociate with (bind to) specific sequences (DNA or other proteins) andalso provides methods for designing and making these miniature proteins.As used herein, the term “miniature protein” or “miniprotein” refers toa relatively small protein containing at least a protein scaffold andone or more additional domains or regions that help to stabilize itstertiary structure. Preferably, these miniature proteins bind to atarget molecule (e.g., DNA or other proteins) with high affinity andselectivity.

As used herein, the term “binding” or “bind to” refers to the specificassociation or other specific interaction between two molecular species,such as protein-protein interactions. It is contemplated that suchassociation is mediated through specific binding sites on each of thetwo interacting molecular species. As used herein, the term “bindingsite” refers to the reactive region or domain of a molecule thatdirectly participates in its specific binding with another molecule. Forexample, when referring to the binding site on a protein, binding occursas a result of the presence of specific amino acid sequence thatinteracts with the other molecule.

Schematically, the invention involves a technique that the inventorshave designated as protein grafting (see, e.g., FIG. 1). In one aspect,this technique identifies critical binding site residues from a proteinthat participate in binding-type association between that protein andits specific binding partners. Then these residues are grafted onto asmall but stable protein scaffold. As used herein, the term “proteinscaffold” refers to a region or domain of a relatively small protein,such as a miniature protein, that has a conserved tertiary structuralmotif which can be modified to display one or more specific amino acidresidues in a fixed conformation. The preferred protein scaffolds of theinvention comprise members of the pancreatic fold (PP fold) proteinfamily, particularly the avian pancreatic polypeptide.

The PP fold protein scaffolds of the invention generally containthirty-six amino acids and are the smallest known globular proteins.Despite their small size, PP fold proteins are stable and remain foldedunder physiological conditions. The preferred PP fold protein scaffoldsof the invention consist of two anti-parallel helices, an N-terminaltype II polyproline helix (PPII) between amino acid residues two andeight, and an alpha-helix between residues 14 and 31 and/or 32. Thestability of the PP fold protein scaffolds of the invention derivespredominantly from interactions between hydrophobic residues on theinterior face of the alpha-helix at positions 17, 20, 24, 27, 28, 30,and 31 and the residues on the two edges of the polyproline helix atpositions 2, 4, 5, 7, and 8. In general, the residues responsible forstabilizing its tertiary structure are not substituted in order tomaintain the tertiary structure of the miniature protein or arecompensated for using phage display.

In certain embodiments, at least one of the critical binding siteresidues of a selected protein is grafted onto the protein scaffold inpositions which are not essential in maintaining tertiary structure,preferably on the type II polyproline helix. In one preferredembodiment, two or three of such binding site residues are grafted ontothe protein scaffold (e.g., aPP). Preferred positions for grafting thesebinding site residues on the protein scaffold include, but are notlimited to, positions on the type II polyproline helix of aPP.Substitutions of binding site residues may be made, although they areless preferred, for residues involved in stabilizing the tertiarystructure of the miniature protein.

A skilled artisan will readily recognize that it is not necessary thatactual substitution of the grafted residues occur on the proteinscaffold. Rather it is necessary that a peptide be identified, through,for example, phage display, that comprises a polypeptide constituting aminiature protein having the association characteristics of the presentinvention. Such peptides may be produced using any conventional means,including, but not limited to synthetic and recombinant techniques.

Members of the PP fold family of protein scaffolds which arecontemplated by the present invention include, but are not limited to,avian pancreatic polypeptide (aPP), Neuropeptide Y, lower intestinalhormone polypeptide, and pancreatic peptide. In the most preferredembodiment, the protein scaffold comprises the PP fold protein, avianpancreatic polypeptide (see, e.g., Blundell et al., 1981, Proc. Natl.Acad. Sci. USA, 78: 4175-4179; Tonan et al., 1990, Biochemistry, 29:4424-4429). aPP is a PP fold polypeptide characterized by a short (eightresidue) amino-terminal type II polyproline helix linked through a typeI beta turn (also referred to herein as the linker region) to aneighteen residue alpha-helix. Because of its small size and stability,aPP is an excellent protein scaffold for, e.g., protein grafting ofpolyproline helix recognition epitopes.

In certain embodiments, the present invention encompasses miniatureproteins that bind to a target protein. Optionally, the binding of theminiature proteins modulates protein-protein interaction between thetarget protein and its binding partner (protein). In one embodiment,making the protein-binding miniature proteins of the invention involvesidentifying the amino acid residues which are essential to binding ofthe target protein to its binding partner. In some embodiments, theseessential residues are identified using three-dimensional models of atarget protein or a protein complex which binds to or interacts withanother protein based on crystallographic studies, while in otherembodiments, they are identified by studies of deletion or substitutionmutants of the target protein. The residues that participate in bindingof the protein to its binding partner are then grafted onto thosepositions which are not necessary to maintain the tertiary structure ofthe protein scaffold to form the protein-binding miniature protein.

The structure of any protein which binds to another protein can be usedto derive the protein-binding miniature proteins of the invention.Preferred embodiments include proline rich sequences on some proteinsthat are folded into type II polyproline (PPII) helices. The PPIIhelical structures can be recognized and bound to by certain proteinmotifs, such as EVH1 domains, SH3 domains, and WW domains.

In a specific embodiment, the invention provides miniature proteins thatbind to a protein of the Ena/VASP family (herein referred to as “anEna/VASP protein”). For example, the protein grafting proceduredescribed herein was applied to the PPII helix of the ActA protein ofListeria monocytogenes to design a miniature protein capable of bindingto an Ena/VASP protein. In this procedure, the primary sequence of aPPII helix of a protein is aligned with residues in the PPII helix ofaPP. Alignments with a large number of conflicts are eliminated as theywould force selection between sequences that were well folded or havehigh affinity, but make it difficult to isolate a molecule with boththese properties. Structural models of the aPP based peptides that areassociated or complexed with the EVH1 domain of an Ena/VASP protein ineach of the alignments are evaluated for unfavorable interactions orsteric clashes between the VanderWaals surface of the Ena/VASP proteinand the backbone of the aPP scaffold. Structural models with multipleunfavorable interactions or steric clashes are eliminated from furtherconsideration.

Examples of the protein-binding miniature proteins which bind to anEna/VASP protein include, but are not limited to, the amino acidsequence depicted in SEQ ID NOs: 1-5 (FIG. 1).

Variants of Miniature Proteins

The miniature proteins of the present invention further includeconservative variants of the miniature proteins herein described. Asused herein, a conservative variant refers to alterations in the aminoacid sequence that do not substantially and adversely affect the bindingor association capacity of the protein. A substitution, insertion ordeletion is said to adversely affect the miniature protein when thealtered sequence prevents or disrupts a function or activity associatedwith the protein. For example, the overall charge, structure orhydrophobic-hydrophilic properties of the miniature protein can bealtered without adversely affecting an activity. Accordingly, the aminoacid sequence can be altered, for example to render the peptide morehydrophobic or hydrophilic, without adversely affecting the activitiesof the miniature protein.

These variants, though possessing a slightly different amino acidsequence than those recited above, will still have the same or similarproperties associated with the miniature proteins depicted in SEQ IDNOs: 1-5. Ordinarily, the conservative substitution variants, will havean amino acid sequence having at least ninety percent amino acidsequence identity with any of the miniature sequences set forth in SEQID NOs: 1-5, more preferably at least ninety-five percent, even morepreferably at least ninety-eight percent, and most preferably at leastninety-nine percent. Identity or homology with respect to such sequencesis defined herein as the percentage of amino acid residues in thecandidate sequence that are identical with the known peptides, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent homology, and not considering any conservativesubstitutions as part of the sequence identity. N-terminal, C-terminalor internal extensions, deletions, or insertions into the peptidesequence shall not be construed as affecting homology.

Thus, the miniature proteins of the present invention include moleculescomprising any of the amino acid sequences of SEQ ID NOs: 1-5; fragmentsthereof having a consecutive sequence of at least about 20, 25, 30, 35or more contiguous amino acid residues of the miniature proteins of theinvention; amino acid sequence variants of such sequences wherein atleast one amino acid residue has been inserted N- or C-terminal to, orwithin, the disclosed sequence; amino acid sequence variants of thedisclosed sequences, or their fragments as defined above, that have beensubstituted by another residue. Contemplated variants further includethose derivatives wherein the protein has been covalently modified bysubstitution, chemical, enzymatic, or other appropriate means with amoiety other than a naturally occurring amino acid (for example, adetectable moiety such as an enzyme or radioisotope).

Nucleic Acid Molecules Encoding Miniature Proteins

The present invention further provides nucleic acid molecules thatencode the subject miniature proteins (e.g., comprising any of the aminoacid sequences of SEQ ID NOs: 1-5) and the related miniature proteinsherein described, preferably in isolated form. As used herein, “nucleicacid” includes cDNA and mRNA, as well as nucleic acids based onalternative backbones or including alternative bases whether derivedfrom natural sources or synthesized. As used herein, a nucleic acidmolecule is said to be “isolated” when the nucleic acid molecule issubstantially separated from contaminant nucleic acid encoding otherpolypeptides from the source of nucleic acid.

The present invention further provides fragments of the encoding nucleicacid molecule. As used herein, a “fragment of an encoding nucleic acidmolecule” refers to a portion of the entire protein encoding sequence ofthe miniature protein. The size of the fragment will be determined bythe intended use. For example, if the fragment is chosen so as to encodean active portion of the protein, the fragment will need to be largeenough to encode the functional region(s) of the protein. Theappropriate size and extent of such fragments can be determinedempirically by persons skilled in the art.

Modifications to the primary structure itself by deletion, addition, oralteration of the amino acids incorporated into the protein sequenceduring translation can be made without destroying the activity of theminiature protein. Such substitutions or other alterations result inminiature proteins having an amino acid sequence encoded by a nucleicacid falling within the contemplated scope of the present invention.

The present invention further provides recombinant DNA molecules thatcontain a coding sequence. As used herein, a recombinant DNA molecule isa DNA molecule that has been subjected to molecular manipulation.Methods for generating recombinant DNA molecules are well known in theart, for example, see Sambrook et al., (1989) Molecular Cloning—ALaboratory Manual, Cold Spring Harbor Laboratory Press. In the preferredrecombinant DNA molecules, a coding DNA sequence is operably linked toexpression control sequences and vector sequences.

The choice of vector and expression control sequences to which one ofthe protein family encoding sequences of the present invention isoperably linked depends directly, as is well known in the art, on thefunctional properties desired (e.g., protein expression, and the hostcell to be transformed). A vector of the present invention may be atleast capable of directing the replication or insertion into the hostchromosome, and preferably also expression, of the structural geneincluded in the recombinant DNA molecule.

Expression control elements that are used for regulating the expressionof an operably linked miniature protein encoding sequence are known inthe art and include, but are not limited to, inducible promoters,constitutive promoters, secretion signals, and other regulatoryelements. Preferably, the inducible promoter is readily controlled, suchas being responsive to a nutrient in the host cell's medium.

In one embodiment, the vector containing a coding nucleic acid moleculewill include a prokaryotic replicon, i.e., a DNA sequence having theability to direct autonomous replication and maintenance of therecombinant DNA molecule extra-chromosomal in a prokaryotic host cell,such as a bacterial host cell, transformed therewith. Such replicons arewell known in the art. In addition, vectors that include a prokaryoticreplicon may also include a gene whose expression confers a detectablemarker such as a drug resistance. Typical of bacterial drug resistancegenes are those that confer resistance to ampicillin or tetracycline.

Vectors that include a prokaryotic replicon can further include aprokaryotic or bacteriophage promoter capable of directing theexpression (transcription and translation) of the coding gene sequencesin a bacterial host cell, such as E. coli. A promoter is an expressioncontrol element formed by a DNA sequence that permits binding of RNApolymerase and transcription to occur. Promoter sequences compatiblewith bacterial hosts are typically provided in plasmid vectorscontaining convenient restriction sites for insertion of a DNA segmentof the present invention. Any suitable prokaryotic host can be used toexpress a recombinant DNA molecule encoding a protein of the invention.

Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can also be used to form a recombinantDNA molecules that contains a coding sequence. Eukaryotic cellexpression vectors are well known in the art and are available fromseveral commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desired DNAsegment.

Eukaryotic cell expression vectors used to construct the recombinant DNAmolecules of the present invention may further include a selectablemarker that is effective in an eukaryotic cell, preferably a drugresistance selection marker. A preferred drug resistance marker is thegene whose expression results in neomycin resistance, i.e., the neomycinphosphotransferase (neo) gene. (Southern et al., (1982) J. Mol. Anal.Genet. 1, 327-341). Alternatively, the selectable marker can be presenton a separate plasmid, the two vectors introduced by co-transfection ofthe host cell, and transfectants selected by culturing in theappropriate drug for the selectable marker.

Transformed Host Cells

The present invention further provides host cells transformed with anucleic acid molecule that encodes a miniature protein of the presentinvention. The host cell can be either prokaryotic or eukaryotic.Eukaryotic cells useful for expression of a miniature protein of theinvention are not limited, so long as the cell line is compatible withcell culture methods and compatible with the propagation of theexpression vector and expression of the gene product.

Transformation of appropriate cell hosts with a recombinant DNA moleculeencoding a miniature protein of the present invention is accomplished bywell known methods that typically depend on the type of vector used andhost system employed. With regard to transformation of prokaryotic hostcells, electroporation and salt treatment methods can be employed (see,for example, Sambrook et al., (1989) Molecular Cloning—A LaboratoryManual, Cold Spring Harbor Laboratory Press; Cohen et al., (1972) Proc.Natl. Acad. Sci. USA 69, 2110-2114). With regard to transformation ofvertebrate cells with vectors containing recombinant DNA,electroporation, cationic lipid or salt treatment methods can beemployed (see, for example, Graham et al., (1973) Virology 52, 456-467;Wigler et al., (1979) Proc. Natl. Acad. Sci. USA 76, 1373-1376).

Successfully transformed cells (cells that contain a recombinant DNAmolecule of the present invention), can be identified by well knowntechniques including the selection for a selectable marker. For example,cells resulting from the introduction of a recombinant DNA of thepresent invention can be cloned to produce single colonies. Cells fromthose colonies can be harvested, lysed and their DNA content examinedfor the presence of the recombinant DNA using a method such as thatdescribed by Southern, (1975) J. Mol. Biol. 98, 503-517 or the proteinsproduced from the cell assayed via an immunological method.

Production of Recombinant Miniature Proteins

The present invention further provides methods for producing a miniatureprotein of the invention using nucleic acid molecules herein described.In general terms, the production of a recombinant form of a proteintypically involves the following steps: a nucleic acid molecule isobtained that encodes a miniature protein of the invention, for example,the nucleic acid molecule encoding the miniature protein depicted in anyof SEQ ID NOs: 1-5. The nucleic acid molecule is then preferably placedin operable linkage with suitable control sequences, as described above,to form an expression unit containing the protein open reading frame.The expression unit is used to transform a suitable host and thetransformed host is cultured under conditions that allow the productionof the recombinant miniature protein. Optionally the recombinantminiature protein is isolated from the medium or from the cells;recovery and purification of the protein may not be necessary in someinstances where some impurities may be tolerated.

Each of the foregoing steps can be done in a variety of ways. Theconstruction of expression vectors that are operable in a variety ofhosts is accomplished using appropriate replicons and control sequences,as set forth above. The control sequences, expression vectors, andtransformation methods are dependent on the type of host cell used toexpress the gene. Suitable restriction sites, if not normally available,can be added to the ends of the coding sequence so as to provide anexcisable gene to insert into these vectors. A skilled artisan canreadily adapt any host/expression system known in the art for use withthe nucleic acid molecules of the invention to produce a recombinantminiature protein.

The present invention further contemplates making the miniature proteinsby chemical synthesis.

Production of Miniature Proteins Using Phase Display

In some embodiments, the present invention contemplates producing andselecting a miniature protein using a phage display method (McCaffertyet al., (1990) Nature 348, 552-554). In this method, display ofrecombinant miniature proteins on the surface of viruses which infectbacteria (bacteriophage or phage) make it possible to produce soluble,recombinant miniature proteins having a wide range of affinities andkinetic characteristics. To display the miniature proteins on thesurface of phage, a synthetic gene encoding the miniature protein isinserted into the gene encoding a phage surface protein (e.g., pIII) andthe recombinant fusion protein is expressed on the phage surface(McCafferty et al., 1990, Nature, 348: 552-554; Hoogenboom et al., 1991,Nucleic Acids Res., 19: 4133-4137). Variability is introduced into thephage display library to select for miniature proteins which not onlymaintain their tertiary, helical structure but which also displayincreased affinity for a preselected target because the critical (orcontributing but not critical) binding residues are optimally positionedon the helical structure.

Since the recombinant proteins on the surface of the phage arefunctional, phage bearing miniature proteins that bind withhigh-affinity to a particular target molecule (e.g., a protein) can beseparated from non-binding or lower affinity phage by antigen affinitychromatography. Mixtures of phage are allowed to bind to the affinitymatrix, non-binding or lower affinity phage are removed by washing, andbound phage are eluted by treatment with acid or alkali. Depending onthe affinity of the miniature protein for its target, enrichment factorsof twenty-fold to a million-fold are obtained by a single round ofaffinity selection. By infecting bacteria with the eluted phage,however, more phage can be grown and subjected to another round ofselection. In this way, an enrichment of a thousand-fold in one roundbecomes a million-fold in two rounds of selection. Thus, even whenenrichments in each round are low (Marks et al., 1991, J. Mol. Biol,222: 581-597), multiple rounds of affinity selection leads to theisolation of rare phage and the genetic material contained within whichencodes the sequence of the domain or motif of the recombinant miniatureprotein that binds or otherwise specifically associates with it bindingtarget.

In various embodiments of the invention, the methods disclosed hereinare used to produce a phage expression library encoding miniatureproteins capable of binding to protein that has already been selectedusing the protein grafting procedure described above. In theseembodiments, phage display can be used to identify miniature proteinsthat display an even higher affinity for a particular target proteinthan that of the miniature proteins produced without the aid of phagedisplay. In yet another embodiment, the invention encompasses auniversal phage display library that can be designed to display acombinatorial set of epitopes or binding sequences to permit therecognition of target molecules (e.g., nucleic acids, proteins or smallmolecules) by a miniature protein without prior knowledge of the naturalepitope or specific binding residues or motifs natively used forrecognition and association.

Various structural modifications are also contemplated for the presentinvention that include the addition of restriction enzyme recognitionsites into the polynucleotide sequence encoding the miniature proteinthat enable genetic manipulation of these gene sequences. Accordingly,the re-engineered miniature proteins can be ligated, for example, intoan M13-derived bacteriophage cloning vector that permits expression of afusion protein on the phage surface. These methods allow for selectingphage clones encoding fusion proteins that bind to a target molecule andcan be completed in a rapid manner allowing for high-throughputscreening of miniature proteins to identify the miniature protein withthe highest affinity and selectivity for a particular target.

According to the methods of the invention, a library of phage displayingmodified miniature proteins is incubated with the immobilized targetmolecule (e.g., a Mena protein) to select phage clones encodingminiature proteins that specifically bind to or otherwise specificallyassociate with the immobilized protein. This procedure involvesimmobilizing a polypeptide sample on a solid substrate. The bound phageare then dissociated from the immobilized polypeptide and amplified bygrowth in bacterial host cells. Individual viral plaques, eachexpressing a different recombinant miniature protein, are expanded toproduce amounts of protein sufficient to perform a binding assay. TheDNA encoding this recombinant binding protein can be subsequentlymodified for ligation into a eukaryotic protein expression vector. Themodified miniature protein, adapted for expression in eukaryotic cells,is ligated into a eukaryotic protein expression vector.

Phage display methods that can be used to make the miniature proteins ofthe present invention include those disclosed in Brinkman et al., (1995)J. Immunol. Methods 182, 41-50; Ames et al., (1995) J. Immunol. Methods184:177-186; Kettleborough et al., (1994) Eur. J. Immunol. 24, 952-958;Persic et al., (1997) Gene 187, 9-18; Burton et al., (1994) Adv.Immunol. 57, 191-280; U.S. Pat. Nos. 5,698,426; 5,223,409; 5,403,484;5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908;5,516,637; 5,780,225; 5,658,727; 5,733,743, 5,837,500 & 5,969,108.

Methods to Identify Binding Partners

In certain embodiments, the present invention relates to methods for usein isolating and identifying binding partners of the miniature proteinsof the invention. In some aspects, a miniature protein of the inventionis mixed with a potential binding partner or an extract or fraction of acell under conditions that allow the association of potential bindingpartners with the miniature protein of the invention. After mixing,peptides, polypeptides, proteins or other molecules that have becomeassociated with a miniature protein of the invention are separated fromthe mixture. The binding partner bound to the protein of the inventioncan then be removed and further analyzed. To identify and isolate abinding partner, the entire miniature protein can be used.Alternatively, a fragment of the miniature protein which contains thebinding domain can be used.

As used herein, a “cellular extract” refers to a preparation or fractionwhich is made from a lysed or disrupted cell. A variety of methods canbe used to obtain an extract of a cell. Cells can be disrupted usingeither physical or chemical disruption methods. Examples of physicaldisruption methods include, but are not limited to, sonication andmechanical shearing. Examples of chemical lysis methods include, but arenot limited to, detergent lysis and enzyme lysis. A skilled artisan canreadily adapt methods for preparing cellular extracts in order to obtainextracts for use in the present methods. Once an extract of a cell isprepared, the extract is mixed with a miniature protein of the inventionunder conditions in which association of the miniature protein with thebinding partner can occur. A variety of conditions can be used, the mostpreferred being conditions that closely resemble conditions found in thecytoplasm of a human cell. Features such as osmolarity, pH, temperature,and the concentration of cellular extract used, can be varied tooptimize the association of the protein with the binding partner.

After mixing under appropriate conditions, the bound complex isseparated from the mixture. A variety of techniques can be utilized toseparate the mixture. For example, antibodies specific to a protein ofthe invention can be used to immunoprecipitate the binding partnercomplex. Alternatively, standard chemical separation techniques such aschromatography and density-sediment centrifugation can be used. Afterremoval of non-associated cellular constituents found in the extract,the binding partner can be dissociated from the complex usingconventional methods. For example, dissociation can be accomplished byaltering the salt concentration or pH of the mixture.

To aid in separating associated binding partner pairs from the mixedextract, the miniature protein of the invention can be immobilized on asolid support. For example, the miniature protein can be attached to anitrocellulose matrix or acrylic beads. Attachment of the miniatureprotein to a solid support aids in separating peptide-binding partnerpairs from other constituents found in the extract. The identifiedbinding partners can be either a single protein or a complex made up oftwo or more proteins. Alternatively, binding partners may be identifiedusing the Alkaline Phosphatase fusion assay according to the proceduresof Flanagan & Vanderhaeghen, (1998) Annu. Rev. Neurosci. 21, 309-345 orTakahashi et al., (1999) Cell 99, 59-69; the Far-Western assay accordingto the procedures of Takayama et al., (1997) Methods Mol. Biol. 69,171-184 or Sauder et al., J. Gen. Virol. (1996) 77, 991-996 oridentified through the use of epitope tagged proteins or GST fusionproteins.

Alternatively, the nucleic acid molecules encoding a miniature proteinof the invention can be used in a yeast two-hybrid system. The yeasttwo-hybrid system has been used to identify other protein partner pairsand can readily be adapted to employ the nucleic acid molecules hereindescribed (see, e.g., Stratagene Hybrizaps two-hybrid system).

EVH1 Domains and Ena/VASP Protein Family

In certain embodiments, miniature proteins of the invention binds to anEna/VASP protein with high affinity and high specificity. Theevolutionarily-conserved Ena/VASP protein family has been implicated inthe regulation of cell migration (Gertler et al., 1996, Cell, 87:227-39). Enabled (Ena) was identified as a genetic suppressor ofloss-of-function mutations in Drosophila Ableson tyrosine kinase (D-Abl)(Gertler et al., 1990, Science, 248: 857-60). Loss-of-function mutationsin Ena ameliorated the embryonic central nervous system defectsassociated with loss of D-Abl in combination with mutations in any ofseveral known D-Abl modifier genes (Gertler, et al., 1995, Genes Dev, 9:521-33). VASP was identified biochemically as an abundant substrate forcyclic-nucleotide dependent kinases in mammalian platelets (Halbrugge etal., 1990, J Chromatogr, 521: 335-43). Two other mammalian members ofthis protein family, Mena (mammalian Enabled) and EVL (Ena/VASP like),were identified by sequence similarity (Gertler et al., 1996, Cell, 87:227-39).

All Ena/VASP family members share a conserved domain structure. TheN-terminal third of the protein, called the EVH1 (Ena VASP Homology)domain, mediates subcellular targeting of Ena/VASP proteins to focaladhesions by binding to proteins containing a motif whose consensus isD/E FPPPPX D/E (Niebuhr et al., 1997, Embo J, 16: 5433-44). Mutationalanalysis indicated that the phenylalanine residue, along with flankingacidic residues on either side, are critical for optimal binding (Carlet al., 1999, Curr Biol, 9: 715-8). The EVH1 ligand motif is found in anumber of cellular proteins, including the focal adhesion proteins zyxinand vinculin. The central portion of Ena/VASP proteins containsproline-rich stretches, which have been reported to be binding sites forthree types of proteins: the G-actin binding protein profilin, SH3domain-containing proteins, and WW domain-containing proteins (Ermekovaet al., 1997, J Biol Chem, 272: 32869-77; Gertler et al., 1996, Cell,87: 227-39). The C-terminal third of Ena/VASP proteins contains the EVH2domain that binds in vitro to F-actin and has a putative coiled-coilregion reported to be important for multimerization (Bachmann et al.,1999, J Biol Chem, 274: 23549-57; Huttelmaier et al., 1999, FEBS Lett,451: 68-74).

In addition to their capacity to bind profilin and actin, thelocalization of Ena/VASP proteins suggests that they may be involved inregulating actin dynamics and/or adhesion (Reinhard et al., 1992, EmboJ., 11: 2063-70; Gertler et al., 1996, Cell, 87: 227-39; Lanier et al.,1999, Neuron, 22: 313-25). Genetic analyses of Ena/VASP family membersin flies and mice demonstrated that these proteins function in processesthat involve cell shape change, and movement including plateletaggregation and axon guidance (Aszodi et al., 1999, Embo J., 18: 37-48;Wills et al., 1999, Neuron, 22: 301-12).

Ena/VASP proteins are also implicated in actin dynamics by their role infacilitating the actin-based motility of the intracellular bacterialpathogen Listeria monocytogenes. The Listeria protein, ActA is requiredfor the formation of actin tails characteristic of motile bacteria(Kocks et al., 1992, Cell, 68: 521-31). Furthermore, the motility of theintracellular pathogen Listeria monocytogenes resulting from rapid actinpolymerization at one pole of the bacterium requires Ena (Laurent etal., 1999, J. Cell Biol, 144: 1245-58; Loisel et al., 1999, Nature 401,613-6). ActA is a multi-domain protein on the surface of the bacteriathat interacts with host cell factors to trigger actin assembly (Pistoret al., 1995, Curr Biol 5: 517-25). Ena/VASP proteins are the only hostcell factors known to bind directly to ActA in vivo, which contains fouroptimized copies of the D/E FPPPPXDDE EVH1 ligand motif (Niebuhr et al.,1997, Embo J., 16: 5433-44). Mutation of these repeats leads to a defectin bacterial movement, despite the fact that an actin cloud and shortactin tails still form around the bacterium (Smith et al., 1996, J. CellBio. 135:647-660; Niebuhr et al., 1997, Embo J, 16: 5433-44).

Therapeutic Uses

The discovery that miniature proteins of the invention display highaffinity for a natural ActA target (e.g., an Ena/VASP protein) and showparalog specificity suggests that miniature proteins can modulatemammalian cell migration. Therefore, these miniature proteins can beimportant therapeutic compounds for diseases such as cancer cellmetastasis, immune regulation, inflammatory disease, andneurodegenerative disorders.

In some aspects of the invention, miniature proteins of the inventionare administrated to a subject in an effective amount to inhibit(completely or partially) migration of a tumor cell across a barrier.The invasion and metastasis of cancer is a complex process whichinvolves changes in cell adhesion properties which allow a transformedcell to invade and migrate through the extracellular matrix (ECM) andacquire anchorage-independent growth properties. Some of these changesoccur at focal adhesions, which are cell/ECM contact points containingmembrane-associated, cytoskeletal, and intracellular signalingmolecules. Metastatic disease occurs when the disseminated foci of tumorcells seed a tissue which supports their growth and propagation, andthis secondary spread of tumor cells is responsible for the morbidityand mortality associated with the majority of cancers. Thus the term“metastasis” as used herein refers to the invasion and migration oftumor cells away from the primary tumor site.

Miniature proteins of the invention are also useful for treating and/orpreventing disorders associated with inflammation in a subject. Forexample, when an Ena/VASP protein activity is induced in immune orhematopoetic cells, the ability of the cells to migrate is reduced.Thus, the subject miniature proteins can induce activity of an Ena/VASPprotein in immune cells such that inflammatory disorders and ischemicdiseases are prevented or treated.

Inflammatory disorders and ischemic diseases are characterized byinflammation associated with neutrophil migration to local tissueregions that have been damaged or have otherwise induced neutrophilmigration and activation. While not intending to be bound by anyparticular theory, it is believed that excessive accumulation ofneutrophils resulting from neutrophil migration to the site of injury,causes the release toxic factors that damage surrounding tissue. Whenthe inflammatory disease is an acute stroke a tissue which is oftendamaged by neutrophil stimulation is the brain. As the activeneutrophils accumulate in the brain an infarct develops.

An “inflammatory disease or condition” as used herein refers to anycondition characterized by local inflammation at a site of injury orinfection and includes autoimmune diseases, certain forms of infectiousinflammatory states, undesirable neutrophil activity characteristic oforgan transplants or other implants and virtually any other conditioncharacterized by unwanted neutrophil accumulation at a local tissuesite. These conditions include but are not limited to meningitis,cerebral edema, arthritis, nephritis, adult respiratory distresssyndrome, pancreatitis, myositis, neuritis, connective tissue diseases,phlebitis, arteritis, vasculitis, allergy, anaphylaxis, ehrlichiosis,gout, organ transplants and/or ulcerative colitis.

An “ischemic disease or condition” as used herein refers to a conditioncharacterized by local inflammation resulting from an interruption inthe blood supply to a tissue due to a blockage or hemorrhage of theblood vessel responsible for supplying blood to the tissue such as isseen for myocardial or cerebral infarction. A cerebral ischemic attackor cerebral ischemia is a form of ischemic condition in which the bloodsupply to the brain is blocked. This interruption in the blood supply tothe brain may result from a variety of causes, including an intrinsicblockage or occlusion of the blood vessel itself, a remotely originatedsource of occlusion, decreased perfusion pressure or increased bloodviscosity resulting in inadequate cerebral blood flow, or a rupturedblood vessel in the subarachnoid space or intracerebral tissue.

The methods of the invention are also useful for treating cerebralischemia. Cerebral ischemia may result in either transient or permanentdeficits and the seriousness of the neurological damage in a patient whohas experienced cerebral ischemia depends on the intensity and durationof the ischemic event. A transient ischemic attack is one in which theblood flow to the brain is interrupted only briefly and causes temporaryneurological deficits, which often are clear in less than 24 hours.Permanent cerebral ischemic attacks, also called stroke, are caused by alonger interruption in blood flow to the brain resulting from either athromboembolism or hemorrhage. A stroke causes a loss of neuronstypically resulting in a neurologic deficit that may improve but thatdoes not entirely resolve.

It has been discovered that mammalian cell migration can be induced bydepleting the cell of functional Ena/VASP protein. Therefore, miniatureproteins of the invention can be useful for regeneration of tissue,including wound healing and neuroregeneration, or prevention ortreatment of neurodegenerative disease.

A “wound” as used herein, means a trauma to any of the tissues of thebody, especially that caused by physical means. The wound healingprocess involves a complex cascade of biochemical and cellular events torestore tissue integrity following an injury. The wound healing processis typically characterized by four stages: 1) hemostasis; 2)inflammation; 3) proliferation; and 4) remodeling. The miniatureproteins of the invention are useful for promoting wound healing bypromoting cellular migration and thus remodeling. In one aspect, themethods of the invention are useful for treating a wound to the dermisor epidermis, e.g., a burn or tissue transplant, injury to the skin.Further, the methods of the invention may be used in the process ofwound healing as well as tissue generation. When the methods of theinvention are used to promote wound healing, cells may be manipulated toalter Ena/VASP activity in vitro and then added to the site of the woundor alternatively the cells present at the site of the wound may bemanipulated in vivo to alter the activity of the Ena/VASP proteins inorder to promote cellular movement. When the methods are used to promotetissue generation, cells can be manipulated and grown in vitro on ascaffold and then implanted into the body or alternatively the scaffoldmay be implanted in the body, or it may be a naturally occurringscaffold and cells manipulated in vivo or in vitro can be used togenerate the tissue.

Another aspect of the invention involves methods for tissueregeneration, which are particularly applicable to growth of neuronalcells. Thus, the invention contemplates the treatment of subjects havingor at risk of developing neurodegenerative disease in order to causeneuroregeneration. Neuronal cells include both central nervous system(CNS) neurons and peripheral nervous system (PNS) neurons. There aremany different neuronal cell types. Examples include, but are notlimited to, sensory and sympathetic neurons, cholinergic neurons, dorsalroot ganglion neurons, and proprioceptive neurons (in the trigeminalmesencephalic nucleus), ciliary ganglion neurons (in the parasympatheticnervous system). A person of ordinary skill in the art will be able toeasily identify neuronal cells and distinguish them from non-neuronalcells such as glial cells, typically utilizing cell-morphologicalcharacteristics, expression of cell-specific markers, and secretion ofcertain molecules.

“Neurodegenerative disorder” is defined herein as a disorder in whichprogressive loss of neurons occurs either in the peripheral nervoussystem or in the central nervous system. Examples of neurodegenerativedisorders include: (i) chronic neurodegenerative diseases such asfamilial and sporadic amyotrophic lateral sclerosis (FALS and ALS,respectively), familial and sporadic Parkinson's disease, Huntington'sdisease, familial and sporadic Alzheimer's disease, multiple sclerosis,olivopontocerebellar atrophy, multiple system atrophy, progressivesupranuclear palsy, diffuse Lewy body disease, corticodentatonigraldegeneration, progressive familial myoclonic epilepsy, strionigraldegeneration, torsion dystonia, familial tremor, Down's Syndrome, Gillesde la Tourette syndrome, Hallervorden-Spatz disease, diabetic peripheralneuropathy, dementia pugilistica, AIDS Dementia, age related dementia,age associated memory impairment, and amyloidosis-relatedneurodegenerative diseases such as those caused by the prion protein(PrP) which is associated with transmissible spongiform encephalopathy(Creutzfeldt-Jakob disease, Gerstmann-Straussler-Scheinker syndrome,scrapie, and kuru), and those caused by excess cystatin C accumulation(hereditary cystatin C angiopathy); and (ii) acute neurodegenerativedisorders such as traumatic brain injury (e.g., surgery-related braininjury), cerebral edema, peripheral nerve damage, spinal cord injury,Leigh's disease, Guillain-Barre syndrome, lysosomal storage disorderssuch as lipofuscinosis, Alper's disease, vertigo as result of CNSdegeneration; pathologies arising with chronic alcohol or drug abuseincluding, for example, the degeneration of neurons in locus coeruleusand cerebellum; pathologies arising with aging including degeneration ofcerebellar neurons and cortical neurons leading to cognitive and motorimpairments; and pathologies arising with chronic amphetamine abuseincluding degeneration of basal ganglia neurons leading to motorimpairments; pathological changes resulting from focal trauma such asstroke, focal ischemia, vascular insufficiency, hypoxic-ischemicencephalopathy, hyperglycemia, hypoglycemia or direct trauma;pathologies arising as a negative side-effect of therapeutic drugs andtreatments (e.g., degeneration of cingulate and entorhinal cortexneurons in response to anticonvulsant doses of antagonists of the NMDAclass of glutamate receptor). and Wemicke-Korsakoff's related dementia.Neurodegenerative diseases affecting sensory neurons includeFriedreich's ataxia, diabetes, peripheral neuropathy, and retinalneuronal degeneration. Neurodegenerative diseases of limbic and corticalsystems include cerebral amyloidosis, Pick's atrophy, and Rettssyndrome. The foregoing examples are not meant to be comprehensive butserve merely as an illustration of the term “neurodegenerativedisorder.”

Miniature proteins of the invention may be administrated to cells of asubject to treat or prevent diseases (e.g., cancer metastasis orinflammatory disorders) alone or in combination with the administrationof other therapeutic compounds for the treatment or prevention of thesedisorders.

Diagnostic Uses

In certain embodiments, miniature proteins of the invention are usefulfor diagnostic purposes to identify the presence and/or detect thelevels of a target protein that binds to the miniature proteins of theinvention. For example, miniature proteins of the invention can be usedto detect the levels of an Ena/VASP protein due to its high affinity andhigh specifity. Miniature proteins of this method can be labeled with adetectable marker. A wide range of detectable markers can be used,including but not limited to biotin, a fluorogen, an enzyme, an epitope,a chromogen, or a radionuclide. The method for detecting the label willdepend on the nature of the label and can be any known in the art, e.g.,film to detect a radionuclide; an enzyme substrate that gives rise to adetectable signal to detect the presence of an enzyme; antibody todetect the presence of an epitope, etc.

In a specific diagnostic embodiment, miniature proteins of the inventionare included in a kit used to detect the presence of a particularprotein (e.g., an Ena/VASP protein) in a biological sample.

Pharmaceutical Compositions.

In certain embodiments, therapeutic compounds of the present invention(e.g., miniature proteins) are formulated with a pharmaceuticallyacceptable carrier. Miniature proteins of the present invention can beadministered alone or as a component of a pharmaceutical formulation(composition). The compounds may be formulated for administration in anyconvenient way for use in human or veterinary medicine. Wetting agents,emulsifiers and lubricants, such as sodium lauryl sulfate and magnesiumstearate, as well as coloring agents, release agents, coating agents,sweetening, flavoring and perfuming agents, preservatives andantioxidants can also be present in the compositions.

Formulations of the miniature proteins include those suitable fororal/nasal, topical, parenteral and/or intravaginal administration. Theformulations may conveniently be presented in unit dosage form and maybe prepared by any methods well known in the art of pharmacy. The amountof active ingredient which can be combined with a carrier material toproduce a single dosage form will vary depending upon the host beingtreated, the particular mode of administration. The amount of activeingredient which can be combined with a carrier material to produce asingle dosage form will generally be that amount of the compound whichproduces a therapeutic effect.

Methods of preparing these formulations or compositions includecombining one compound and a carrier and, optionally, one or moreaccessory ingredients. In general, the formulations are prepared bycombining a compound with a liquid carrier, or a finely divided solidcarrier, or both, and then, if necessary, shaping the product.

Formulations of the miniature proteins suitable for oral administrationmay be in the form of capsules, cachets, pills, tablets, lozenges (usinga flavored basis, usually sucrose and acacia or tragacanth), powders,granules, or as a solution or a suspension in an aqueous or non-aqueousliquid, or as an oil-in-water or water-in-oil liquid emulsion, or as anelixir or syrup, or as pastilles (using an inert base, such as gelatinand glycerin, or sucrose and acacia) and/or as mouth washes and thelike, each containing a predetermined amount of a compound as an activeingredient. A compound may also be administered as a bolus, electuary orpaste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), a miniature protein is mixedwith one or more pharmaceutically acceptable carriers, such as sodiumcitrate or dicalcium phosphate, and/or any of the following: (1) fillersor extenders, such as starches, lactose, sucrose, glucose, mannitol,and/or silicic acid; (2) binders, such as, for example,carboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose, and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and sodium carbonate;(5) solution retarding agents, such as paraffin; (6) absorptionaccelerators, such as quaternary ammonium compounds; (7) wetting agents,such as, for example, cetyl alcohol and glycerol monostearate; (8)absorbents, such as kaolin and bentonite clay; (9) lubricants, such atalc, calcium stearate, magnesium stearate, solid polyethylene glycols,sodium lauryl sulfate, and mixtures thereof; and (10) coloring agents.In the case of capsules, tablets and pills, the pharmaceuticalcompositions may also comprise buffering agents. Solid compositions of asimilar type may also be employed as fillers in soft and hard-filledgelatin capsules using such excipients as lactose or milk sugars, aswell as high molecular weight polyethylene glycols and the like.

Liquid dosage forms for oral administration of a miniature proteininclude pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups, and elixirs. In addition to the activeingredient, the liquid dosage forms may contain inert diluents commonlyused in the art, such as water or other solvents, solubilizing agentsand emulsifiers, such as ethyl alcohol, isopropyl alcohol, ethylcarbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-butylene glycol, oils (in particular, cottonseed, groundnut,corn, germ, olive, castor, and sesame oils), glycerol, tetrahydrofurylalcohol, polyethylene glycols and fatty acid esters of sorbitan, andmixtures thereof. Besides inert diluents, the oral compositions can alsoinclude adjuvants such as wetting agents, emulsifying and suspendingagents, sweetening, flavoring, coloring, perfuming, and preservativeagents.

Suspensions, in addition to the active compounds (e.g., miniatureproteins), may contain suspending agents such as ethoxylated isostearylalcohols, polyoxyethylene sorbitol, and sorbitan esters,microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agarand tragacanth, and mixtures thereof.

Methods of the invention can be administered topically, either to skinor to mucosal membranes (e.g., those on the cervix and vagina). Thisoffers the greatest opportunity for direct delivery to tumor with thelowest chance of inducing side effects. The topical formulations mayfurther include one or more of the wide variety of agents known to beeffective as skin or stratum corneum penetration enhancers. Examples ofthese are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide,dimethylformamide, propylene glycol, methyl or isopropyl alcohol,dimethyl sulfoxide, and azone. Additional agents may further be includedto make the formulation cosmetically acceptable. Examples of these arefats, waxes, oils, dyes, fragrances, preservatives, stabilizers, andsurface active agents. Keratolytic agents such as those known in the artmay also be included. Examples are salicylic acid and sulfur.

Dosage forms for the topical or transdermal administration of a compound(e.g., a miniature protein) include powders, sprays, ointments, pastes,creams, lotions, gels, solutions, patches, and inhalants. The activecompound may be mixed under sterile conditions with a pharmaceuticallyacceptable carrier, and with any preservatives, buffers, or propellantswhich may be required. The ointments, pastes, creams and gels maycontain, in addition to a therapeutic compound, excipients, such asanimal and vegetable fats, oils, waxes, paraffins, starch, tragacanth,cellulose derivatives, polyethylene glycols, silicones, bentonites,silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a compound, excipientssuch as lactose, talc, silicic acid, aluminum hydroxide, calciumsilicates, and polyamide powder, or mixtures of these substances. Sprayscan additionally contain customary propellants, such aschlorofluorohydrocarbons and volatile unsubstituted hydrocarbons, suchas butane and propane.

Pharmaceutical compositions suitable for parenteral administration maycomprise one or more compounds in combination with one or morepharmaceutically acceptable sterile isotonic aqueous or nonaqueoussolutions, dispersions, suspensions or emulsions, or sterile powderswhich may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with theblood of the intended recipient or suspending or thickening agents.Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions of the invention includewater, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof,vegetable oils, such as olive oil, and injectable organic esters, suchas ethyl oleate. Proper fluidity can be maintained, for example, by theuse of coating materials, such as lecithin, by the maintenance of therequired particle size in the case of dispersions, and by the use ofsurfactants.

Injectable depot forms are made by forming microencapsule matrices ofthe compounds in biodegradable polymers such aspolylactide-polyglycolide. Depending on the ratio of drug to polymer,and the nature of the particular polymer employed, the rate of drugrelease can be controlled. Examples of other biodegradable polymersinclude poly(orthoesters) and poly(anhydrides). Depot injectableformulations are also prepared by entrapping the drug in liposomes ormicroemulsions which are compatible with body tissue.

Formulations of the compounds for intravaginal administration may bepresented as a suppository, which may be prepared by mixing one or morecompounds of the invention with one or more suitable nonirritatingexcipients or carriers comprising, for example, cocoa butter,polyethylene glycol, a suppository wax or a salicylate, and which issolid at room temperature, but liquid at body temperature and,therefore, will melt in the rectum or vaginal cavity and release theactive compound. Optionally, such formulations suitable for vaginaladministration also include pessaries, tampons, creams, gels, pastes,foams or spray formulations containing such carriers as are known in theart to be appropriate.

Exemplification

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Introduction

EVH1 domains are found within a large number of multi-domain signalingproteins that regulate the dynamics of the actin cytoskeleton, includingthose where external stimuli regulate cellular motility, shape, andadhesion (1). Examples include Drosophilia Enabled (Ena) (2) and itsmammalian counterparts Mena (1a), vasodilator-stimulated phosphoprotein(VASP) (3), Enabled/VASP-like protein (Evl) (1a), and Wiskott-Aldrichsyndrome protein (WASP) (4). EVH1 domains regulate actin filamentdynamics through interactions with cytoskeleton-associated proteinsincluding vinculin and zyxin, and are used by the ActA protein ofListeria monocytogenes during pathogenesis (5). Like SH3 and WW domains,EVH1 domains recognize proline-rich sequences on target proteins (6)that re folded into type II polyproline (PPII) helices (7). In the caseof L. monocytogenes, the interaction of intracellular EVH1 domains withActA contribute to the propulsion of the bacterium through the host cellcytoplasm and into neighboring cells (8).

Results

Previously, we have described a miniature protein design strategy inwhich the well-folded helix in avian pancreatic polypeptide (aPP)presents short α-helical recognition epitopes (FIG. 1A) (10, 11). Theminiature proteins so designed recognize even shallow clefts on proteinsurfaces with nanomolar affinities and high specificity (11). aPPconsists of an eight-residue PPII helix linked through a type I β-turnto a 20 residue α-helix. Here we extend this protein design strategy tostabilize the functional epitope of ActA on the PPII helix of aPP. Likeminiature proteins that use an α-helix for protein recognition, theminiature protein designed in this way displays high affinity for theMena₁₋₁₁₂ EVH1 domain and achieves the elusive goal of paralogspecificity (12) discriminating well between EVH1 domains of Mena₁₋₁₁₂,VASP₁₋₁₁₅ and Evl₁₋₁₁₂.

Our design began with the structure of Mena₁₋₁₁₂ in complex with theproline-rich peptide F₁P₂PP₄P₅ (FP₄) (13). The structure shows thepentapeptide bound as a type II polyproline helix, with residues P₂, P₄and P₅ nestled into the concave, V-shaped, binding surface on Mena₁₋₁₁₂,and residue F₁ anchoring the peptide in the N to C direction (13).Substitution of FP₄ residues F₁, P₂, and P₅ at positions S₃, Q₄, and Y₇and aPP, and extension of this core sequence by two of three C-terminalacidic residues shown to improve affinity and specificity (13, 14, 5c)led to the final sequence of pGolemi (FIG. 1B).

pGolemi was synthesized using standard solid phase methods (9) andexamined by circular dichroism (CD) spectroscopy (FIG. 1C). The CDspectrum of pGolemi at 25° C. exhibited minima at 208 and 222 nm, asexpected for a protein containing one or more α-helices, and wasindependent of concentration between 5 and 20 μM. The mean residueellipticity (Θ_(MRE)) at 222 nm of −5,500 deg·cm²·dmol⁻¹ suggests thatapproximately 60% of pGolemi possessed an α-helical conformation. Thestability of pGolemi was examined further by monitoring thetemperature-dependence of Θ_(MRE) at 222 nm. pGolemi underwent areversible, moderately cooperative melting transition with T_(m)=50° C.(FIG. 1D). These data suggest that pGolemi adopts a stable, folded,monomeric, aPP-like structure.

The affinity and specificity of pGolemi.EVH1 domain interactions weremeasured by tryptophan perturbations analysis (FIG. 2A) (13). An 11residue peptide comprising PPII repeat 2 of L. monocytogenenes ActA(ActA₁₁) and two peptides comprising the N-terminal 7 or 11 residues inpGolemi (PPII₇ and PPII₁₁) were prepared as controls. pGolemi boundMena₁₋₁₁₂ with high affinity (K_(d)=700±30 nM) (9). This affinity is10-fold higher than that of ActA₁₁, the best previously known Menaligand (13). The interaction between pGolemi and Mena₁₋₁₁₂ was confirmedby fluorescence polarization experiments using a fluorescent pGolemiderivative (pGolemi^(Flu)) (FIG. 2B); the K_(d) determined this way was290±50 nM. Furthermore, pGolemi and ActA₁₁ compete with pGolemi^(Flu)for binding to Mena₁₋₁₁₂ with IC₅₀ values of 542±30 nM and 4.0±0.2 μM,respectively (9). Interestingly, PPII₇ and PPII₁₁ were poor Mena₁₋₁₁₂ligands (K_(d)=480 μM and >1 mM, respectively), indicating that thepGolemi α-helix contributes at least 3.5 kcal·mol⁻¹ to the Mena₁₋₁₁₂affinity of pGolemi.

The folded structure of pGolemi also contributes to its ability todifferentiate EVH1 domain paralogs (FIG. 2C). The sequences of EHV1domains Mena₁₋₁₁₂, VASP₁₋₁₁₅, and Evl₁₋₁₁₅ are 60% identical and theirstructures are virtually superimposable (14). Although ActA₁₁ bindsequally to all EVH1 domains tested (FIG. 2D, K_(rel)<3) pGolemi prefersMena₁₋₁₁₂ to VASP₁₋₁₁₅ (K_(re1)=20) and especially to Evl₁₋₁₁₅(K_(rel)>120) (FIG. 2C). This level of specificity (K_(rel) of 66and >345, respectively) was confirmed by fluorescence polarizationanalysis (FIG. 2B). pGolemi also discriminated well between Mena₁₋₁₁₂and protein domains that recognize proline-rich sequences or α-helices;its affinity for the KIX domain of CBP, which recognizes α-helicalligands, was 15±0.7 μM; no interaction was detected between pGolemi andthe N- or C-terminal SH3 domains of Grb-2 (9).

The properties of pGolemi were also examined in Xenopus laevis eggcytoplasmic extracts that reconstitute L. monocytogenes actin-basedmotility (FIG. 3) (15). L. monocytogenes motility in mammalian cells andextracts is due to interactions between the 639-residue bacterialprotein ActA and host proteins that recruit and activate actinpolymerization. Addition of 10 μM ActA₁₁ decreased the median speed ofL. monocytogenes by 89%, consistent with previous work. (16, 5b)Addition of 10 or 27 μM pGolemi decreased the median speed of L.monocytogenes by 68% (FIG. 3A), but in addition caused extreme speedvariations and discontinuous tail formation at all times (FIG. 3C).Discontinuous tails were not observed at any concentration of ActA₁₁tested (FIG. 3D). The differential effects of ActA₁₁ and pGolemi on L.monocytogenes motility may reflect their different specificities amongEVH1 domain family members. Further experimentation will be required tofully understand the molecular events that result in altered motility.

Many protein-protein interactions in cell signaling demand interactionswith proline rich sequences (6), and the design of molecules thatperturb signaling pathways represents a foremost goal of chemicalbiology. Our results suggest that miniature proteins based on aPP mayrepresent an excellent framework for the design of ligands thatdifferentiate the roles of EVH1 domains in vitro and in vivo.

REFERENCES

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INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject invention have been discussed,the above specification is illustrative and not restrictive. Manyvariations of the invention will become apparent to those skilled in theart upon review of this specification and the claims below. The fullscope of the invention should be determined by reference to the claims,along with their full scope of equivalents, and the specification, alongwith such variations.

1. An avian pancreatic polypeptide (aPP) modified by substitution of atleast one amino acid residue, wherein said at least one residue islocated within a type II polyproline (PPII) helix of the polypeptidewhen the polypeptide is in a tertiary form.
 2. The modified polypeptideof claim 1, wherein at least two or three amino acid residues on thePPII helix are substituted.
 3. The modified polypeptide of claim 1,further modified by substitution of at least one amino acid residue inthe linker region between the PPII helix and the alpha helix domain ofthe polypeptide.
 4. The modified polypeptide of claim 3, wherein atleast two amino acid residues on the linker region are substituted. 5.The modified polypeptide of claim 1, wherein said at least onesubstituted residue of the PPII helix is selected from a site on a firstprotein through which the first protein interacts with a second protein.6. The modified polypeptide of claim 5, wherein the first protein isselected from the group consisting of: zyxin, vinculin, and the ActAprotein of Listeria monocytogenes.
 7. The modified polypeptide of claim5, wherein the second protein is selected from the group consisting of:Drosophila Enabled (Ena), mammalian Mena, vasodilator stimulatedphosphoprotein (VASP), Enabled/VASP-like protein (Evl), andWiskott-Aldrich syndrome protein (WASP).
 8. The modified polypeptide ofclaim 5, wherein the site is a protein binding site.
 9. The modifiedpolypeptide of claim 1, having an amino acid sequence selected from anyof SEQ ID NOs: 1-5.
 10. The modified polypeptide of claim 1, wherein themodified polypeptide binds to a protein selected from the groupconsisting of: Drosophila Enabled (Ena), mammalian Mena, vasodilatorstimulated phosphoprotein (VASP), Enabled/VASP-like protein (Evl), andWiskott-Aldrich syndrome protein (WASP).
 11. The modified polypeptide ofclaim 10, wherein the modified polypeptide preferentially binds to oneprotein selected from the group consisting of: Drosophila Enabled (Ena),mammalian Mena, vasodilator stimulated phosphoprotein (VASP),Enabled/VASP-like protein (Evl), and Wiskott-Aldrich syndrome protein(WASP), but doe not bind to the other proteins of the group.
 12. Themodified polypeptide of claim 1, wherein the modified polypeptideinhibits interaction between the known protein and the second protein.13-17. (canceled)
 18. An isolated polypeptide selected from the groupconsisting of: (a) an isolated polypeptide comprising any of the aminoacid sequences as set forth in SEQ ID NOs: 1-5; (b) an isolatedpolypeptide comprising a fragment of at least twelve contiguous aminoacids of any of SEQ ID NOs: 1-5; (c) an isolated polypeptide comprisingone or more amino acid substitutions in any of the amino acid sequencesas set forth in SEQ ID NOs: 1-5; and (d) an isolated polypeptide atleast 95 percent identical to any of SEQ ID NOs: 1-5. 19-20. (canceled)21. A method of identifying an avian pancreatic polypeptide (aPP)miniature protein that modulates the interaction between a known proteinand a second protein, comprising a step of isolating at least onerecombinant phage clone from a phage display library that displays aprotein scaffold that modulates the association between a known proteinand a second protein, the phage display library comprising a pluralityof recombinant phase that express a protein scaffold modified bysubstitution of at least one amino acid residue, said at least oneresidue being exposed on a type II polyproline (PPII) helix of thepolypeptide when the polypeptide is in a tertiary form. 22-38.(canceled)